The present invention relates to a monoclonal antibody recognizing amyloid A in human serum (hereinafter abbreviated as SAA) and cause the agglutination reaction, a reagent for immunoassay comprising the monoclonal antibody, and a method for immunoassay using the monoclonal antibody.
In such fields as clinical examinations, immunoassay is often utilized in order to measure certain substances simply and conveniently or with high sensitivity and specificity. Antibodies are required for immunoassay. Monoclonal antibodies are indispensable tools for immunoassay because of their numerous advantages: that they can be continuously supplied with uniform characteristics; that an amount of antigens used can be small since the antibody-producing cells can be established as cell lines; and among others, that antibodies of high specificity can be easily obtained. Monoclonal antibodies are important tools not only for assay but also for purification of substances and for studies on their physiological activities and in vivo behavior.
SAA is a serum protein with a molecular weight of about 12,000, which is considered as a precursor protein of amyloid protein A (hereinafter abbreviated as AA protein) that is deposited in tissues in a certain type of amyloidosis (J. Clin. Invest. 53: 1054-1061, 1974). Recently, it was reported that the serum SAA value rises in inflammatory disorders, and therefore it has been recognized as a sensitive marker for inflammations (Rinshokensa 32: 2, p 168, 1988).
There are some reports on the monoclonal antibodies recognizing SAA (J. Immunol. Methods 144, 149-155, 1991; Clin. Chem. 40/7, 1284-1290, 1994). In these reports, ELISA is constituted with the monoclonal antibodies recognizing SAA.
Generally speaking, those monoclonal antibodies that are sufficiently usable in ELISA often are not practically useful in the agglutination method. This is because the antibody to be used must have the following characteristics in either case of utilizing carrier particles like latex or of not requiring a carrier as in the case of immunological turbidimetric analysis.
First of all, the agglutination method requires higher affinity. Compared to ELISA, it requires generally a higher level of affinity in order to achieve the agglutination reaction and to maintain a physically stable aggregate. Especially with monoclonal antibodies, one would need to prepare those with high affinities because the reaction needs to be constituted solely with the antibody molecules that react specifically with certain epitopes. However, conventional monoclonal antibodies cannot satisfy this requirement. If one attempts to constitute a reaction system with a monoclonal antibody with insufficient affinity, it is impossible to conduct measurement by the agglutination method because the antibody does not form an aggregate that can be analyzed with practical sensitivity. If the quantity of the antibody is increased to compensate for the low affinity, sufficient sensitivity cannot be obtained since the number of antibody molecules that can bind to one antigen molecule does not change. The measure to increase the quantity of the antibody is also unsatisfactory when using carriers such as latex by binding it to the antibody because the amount of the antibody bound is limited.
Furthermore, the agglutination reaction requires multiple existence of the epitope recognized by the monoclonal antibody on a single antigen and positional relationship among the epitope at multiple sites must be suitable for agglutination. Therefore, the positional relationship, which is not an issue in ELISA, may become an obstacle in the agglutination method. Although the same condition is required in the sandwich ELISA method, it is disadvantageous to rely solely on the contiguous epitopes even if their physical sites are different because, as described earlier, the agglutination method requires a physically stronger binding. This is because steric hindrance tends to occur, which makes it difficult to obtain a large, stable aggregate. Thus, also from the standpoint of epitope selection, conventional monoclonal antibodies are not suitable for the agglutination method.
An object of the present invention is to provide a novel monoclonal antibody that enables measurement of SAA based on the agglutination reaction. Another object of the invention is to provide a novel reagent for immunoassay comprising the monoclonal antibody, and a novel method for immunoassay using the monoclonal antibody.
Using highly purified SAA and enhancing the antigenicity of SAA by combining SAA with various adjuvant components to make it into a unique form of immunogen, the present inventors have obtained several kinds of hybridomas producing the monoclonal antibodies recognizing human SAA with the following reactivity characteristics: (1) that they recognize the epitope of human serum amyloid A, and (2) that they agglutinate by reacting with human serum amyloid A in the absence/presence of other monoclonal antibodies, to isolate several monoclonal antibodies from the hybridomas. The present inventors also conducted agglutination experiments of SAA using the monoclonal antibodies, and have established a novel method for immunoassay utilizing the monoclonal antibodies, thereby completing the invention.
The monoclonal antibodies of the present invention enable immunoassay by the agglutination reaction. The use of currently available monoclonal antibodies against SAA has been limited to such measurement methods as ELISA because they have insufficient affinities for the agglutination reaction. Since the monoclonal antibodies of the present invention have new characteristics that they agglutinate by reacting with SAA, it is possible to provide a convenient method for measuring SAA based on the agglutination reaction.
The monoclonal antibodies of the present invention can be obtained by immunizing mice, rats, etc. with the purified SAA, and immortalizing the antibody-producing cells by some means. As to the techniques for immortalization, known techniques include cell fusion with tumor cells such as myelomas and transformation with Epstein-Barr virus.
When preparing hybridomas by cell fusion, the myeloma cells from the same animal species as the immunized animal may be used, or they can originate from some other animals, thereby creating hetero-hybridoma cells. SAA is a protein also found in serum of non-human animals, and it increases by immunization as a stimulus. Consequently it is advantageous to use an animal species that does not show such a phenomenon, e.g., rats, as the animal to be immunized because the antibody titer is likely to rise.
SAA to be utilized as the immunogen can be obtained by purification using a known method. Specifically, SAA can be recovered as a pure protein by obtaining a high density lipoprotein (hereinafter abbreviated as HDL) fraction from crude serum by ultracentrifugation, subjecting it to delipidation, and purifying by means of, e.g., ion exchange chromatography. It is desirable to use such highly purified protein in order to obtain the cells that produce monoclonal antibodies specific to SAA in a high yield. Although there is a report that the unpurified HDL fraction obtained through ultracentrifugation was used as an immunogen, the antibody-producing cells obtained using such immunogens often produce antibodies recognizing various apolipoprotein antigens, which may be disadvantageous in terms of, e.g., cloning.
When using purified SAA as an immunogen, a variety of techniques can be applied to enhance its antigenicity. Freund""s complete adjuvant (hereinafter abbreviated as FCA) is one of the necessary components to enhance immunogenicity. It is also effective to use the SAA adsorbed to lipid liposomes to serve as an immunogen. It is pointed out that SAA exhibits poor antigenicity in the immunized animals since SAA is a protein inherently found in many mammals"" blood. One of the reasons why there is no report on the monoclonal antibody usable in the agglutination reaction is presumed to be this low antigenicity of SAA, and therefore it is important to enhance the antigenicity in order to obtain the monoclonal antibody of the present invention. An adjuvant component useful in obtaining the monoclonal antibody of the present invention is exemplified by FCA supplemented with cells of tubercle bacilli. FCA originally contains tubercle bacilli, but better results can be expected by reinforcing this component. An intramuscular injection of the pertussis vaccine at the time of immunization can also be expected to enhance the immunization effects.
In the present invention, agglutination reaction of the monoclonal antibody in response to SAA can be confirmed through the following method. The simplest method may be to allow the monoclonal antibody and SAA to react under a favorable condition for the immune reaction and to confirm the agglutination ability by observing as an indicator as to whether the immune complex precipitation forms. By this method, however, it is generally difficult to perform a quantitative observation without using a sensitizing agent or the like.
It is preferable to use the latex agglutination reaction in order to quantitatively measure agglutination ability of the monoclonal antibody. An example of the method for confirming the agglutination ability of the monoclonal antibody by the latex agglutination reaction is described below.
The anti-SAA monoclonal antibody is physically adsorbed to polystyrene latex (average particle diameter 0.109 xcexcm) at 37xc2x0 C. for 1 hour, and then suspended in a dispersion medium (0.1 M HEPES buffer, pH 7.4, containing 1% BSA) to give the final latex concentration of 1%, resulting in a monoclonal antibody-sensitized latex emulsion. This emulsion is reacted with a sample having a certain SAA concentration, and the resulting agglutination is optically measured. Known optical measurement methods include the method of measuring changes in absorbance and changes in light scattering.
The following is an example of the measurement of absorbance changes using the Immunological Latex Agglutination Reaction Measurement System LA-2000 (trade name, manufactured by EIKEN KAGAKU-Analytical Instrument). The measurement parameters are shown below. With these parameters, the absorbance changes over 400 seconds after the initiation of reaction are calculated as DOS. DOS is a specific value to LA-2000.
Under these reaction conditions, 0.1 M HEPES buffer (pH 7.4) alone is measured in order to determine the optical fluctuation of the instrument itself. Then, as a blank test, the same buffer is used as a sample to obtain the reagent blank. The basic value (lower limit) is calculated by adding, to the reagent blank average, its doubled standard deviation and further the instrument fluctuation range (reagent blank+2 SD+instrument""s fluctuation range). Occurrence of agglutination can be judged when a measured value is larger than the basic value. Similar test methods can be performed using a general spectrophotometer, without using specialized systems such as LA-2000.
The monoclonal antibodies of the present invention include those that react with SAA by themselves to cause agglutination, and those that react with SAA in the presence of various anti-SAA monoclonal antibodies to cause agglutination. Moreover, among those that react with SAA by themselves to cause the agglutination reaction, there are the antibodies that exhibit a stronger agglutination in the presence of various anti-SAA monoclonal antibodies than that attainable when they are used individually.
For example, the following data were obtained by measuring the binding affinities of the monoclonal antibodies shown in FIG. 1. Since there were no notable correlation between the affinity constant and the intensity of agglutination activity, it seemed impossible to simply infer the strength of agglutination activity of the monoclonal antibody to SAA based on its affinity.
Clone 15: 8.4xc3x9710xe2x88x927 
Clone 16: 2.0xc3x9710xe2x88x928 
Clone 17: 1.0xc3x9710xe2x88x927 
Clone 18: 8.5xc3x9710xe2x88x927 
Furthermore, while the monoclonal antibodies, which was actually obtained by the present inventors, recognized the regions shown, for example in FIG. 4, as epitopes, there were no correlation between the epitopes and the agglutination activities. The epitopes shown in FIG. 4 were obtained by analyzing the representative monoclonal antibodies among those described in FIG. 1. Therefore, it seems difficult to predict the agglutination activity of the monoclonal antibodies recognizing SAA based solely on the epitopes. The names of amino acids are herein abbreviated as follows.
The hybridomas SAA-17 and SAA-21, obtained by the present inventors and included in the present invention, have been respectively deposited under the following conditions.
Deposit concerning hybridoma SAA-17:
(A) Name and address of depositary institution
Name: National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry
Address: 1-3 Higashi 1-chome, Tsukuba-shi, Ibaragi, Japan (postal code 305)
(B) Date of deposit (Original date of deposit) Aug. 2, 1995
(C) Accession number FERM BP-5616
Deposit concerning hybridoma SAA-21:
(A) Name and address of depositary institution
Name: National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry
Address: 1-3 Higashi 1-chome, Tsukuba-shi, Ibaragi, Japan (postal code 305)
(B) Date of deposit (Original date of deposit) Aug. 2, 1995
(C) Accession number FERM BP-5617
These hybridomas are rat-mouse heterohybridomas established by fusing the antibody-producing cells derived from the rat, which was immunized using the method described above, with the mouse myeloma cells. These hybridomas can be injected into the abdominal cavity of nude mice to recover the ascites and to purify the monoclonal antibodies. They can also be cultured in an appropriate medium to obtain the culture supernatant and to purify the monoclonal antibodies.
The monoclonal antibodies of the present invention exhibit differences in agglutination characteristics, as described earlier, depending on the combination when plural antibodies are used. Therefore, when utilizing these monoclonal antibodies as reagents, one should empirically choose appropriate combinations to readily achieve the expected sensitivity and the range of measurement. The number of monoclonal antibodies to be combined is not limited to two, but larger numbers of monoclonal antibodies can be combined. An aggregate that exhibits more stable and stronger binding can be expected to be formed when three or more different kinds of monoclonal antibodies are used in combination. Alternatively, it is also possible to control the agglutination intensity or the quantifiable range by manipulating the combination and the mixing ratio.
When the reagent of the present invention is prepared by coupling plural monoclonal antibodies to the carrier particles, it can be prepared by either the method of mixing after each species of monoclonal antibody is coupled to the carrier particle or the method of coupling the mixture of plural monoclonal antibodies. The former method may be advantageous because one can accommodate the subtle differences in binding conditions among different clones and it is easy to adjust the mixing ratio of antibodies.
As the reagent for immunoassay of the present invention, the monoclonal antibody can be used in a free state or as coupled to an insoluble carrier. Preferable insoluble carriers include particulate carriers made of synthetic organic materials such as latex, and inorganic materials such as silica, alumina, gold colloid, or the like. In order to couple the monoclonal antibody to these particulate carriers, chemical bonding or physical adsorption may be utilized.
The monoclonal antibodies that constitute the reagents of the present invention can be utilized as fragments resulted from digestion with appropriate enzymes for the purpose of suppressing non-specific influences of the rheumatoid factor and complements. Known antibody fragments include F(abxe2x80x2)2 given by pepsin digestion, Fab by papain digestion, and Facbxe2x80x2 by plasmin digestion.
It is possible to combine known ingredients other than those mentioned above with the reagent for immunoassay of SAA according to the present invention. Such ingredients include buffers that provide necessary pH for immune reactions, reaction enhancers that promote immune reactions, reaction stabilizers or blockers that suppress non-specific reactions, and preservatives such as sodium azide that improve preservability of the reagent.
Among the buffers applicable in the present invention, GOOD""s buffers are particularly preferred because they not only provide the advantageous pH for immune reactions but also have minimum influence on proteins. Followings are used as GOOD""s buffers.
2-(N-Morpholino)ethanesulfonic acid (abbreviated as MES)
Piperazine-N,Nxe2x80x2-bis(2-ethane sulfonic acid) (abbreviated as PIPES)
N-(2-Acetamido)-2-aminoethanesulfonic acid (abbreviated as ACES)
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (abbreviated as BES)
Bis(2-hydoxyethyl)iminotris(hydroxymethyl)methane (abbreviated as Bis-Tris)
3-[N,N-Bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (abbreviated as DISPO)
N-2-Hydroxyethylpiperazine-Nxe2x80x2-3-propanesulfonic acid (abbreviated as EPPS)
N-2-Hydroxyethylpiperazine-Nxe2x80x2-2-ethanesulfonic acid (abbreviated as HEPES)
N-2-Hydroxyethylpiperazine-Nxe2x80x2-2-hydroxypropane-3-sulfonic acid (abbreviated as HEPPSO)
3-(N-Morpholino)propanesulfonic acid (abbreviated as MOPS)
3-(N-Morpholino)-2-hydroxypropanesulfonic acid (abbreviated as MOPSO)
Piperazine-N,Nxe2x80x2-bis(2-hydroxypropanesulfonic acid) (abbreviated as POPSO)
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (abbreviated as TAPS)
N-Tris(hydroxymethyl)methyl-2-hydroxy-3-aminopropane-sulfonic acid (abbreviated as TAPSO)
N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (abbreviated as TES)
Other buffers, such as the following, may also be used.
2-Amino-2-hydroxymethyl-1,3-propanediol, also called Tris(hydroxymethyl)aminomethane
Phosphate buffers
Ammonium buffers
The reaction enhancers include polyethyleneglycol and dextran sulfate. The reaction stabilizers and blockers usable in the present invention include BSA (bovine serum albumin), animal sera, IgG, IgG fragments (Fab or Fc), albumins, milk proteins, amino acids, poly-amino acids, choline, polysaccharides such as sucrose, gelatin, decomposition products of gelatin, casein, and polyhydric alcohols such as glycerol, which are effective for stabilizing the reaction or suppressing nonspecific reactions during the immune reaction.
The reagent for immunoassay of SAA of the present invention, which may include the various components as mentioned above, can be provided as solutions or in a dry state. In providing them as solutions, materials such as surfactants, carbohydrates, and inactive proteins can be further added in order to enhance the stability of the proteins. These stabilizing agents are also effective as stabilizers or vehicles when the reagents are dried.
The immunoassay of SAA according to the present invention are performed using the reagent for immunoassay comprising the monoclonal antibodies of the present invention as described above. The immunoassay can be carried out by adding the reagents to the sample and monitoring the progress of the agglutination reaction optically or with the naked eyes.